PGE2 belongs to the family of prostaglandins (PGs), which are a group of biologically active compounds found in virtually all tissues and organs. They are synthesized from the metabolism of a membrane lipid, arachidonic acid, by cyclooxygenases to the intermediate PGH2, which then serves as the substrate for generation of five prostanoids: PGE2, PGF2, PGD2, PGI2 (prostacyclin), and TxA2 (thromboxane A2) (Romanovsky et al. (2005) Fever and hypothermia in systemic inflammation: recent discoveries and revisions. Front Biosci. 10:2193-2216). PGs have a variety of functions in the human body including regulation of blood pressure, blood clotting, and sleep. However, they also play a major role in the mediation and modulation of pain and inflammation, and are therefore targets of NSAIDs (non-steroid anti-inflammatory drugs).
PGE2 is synthesized by various enzymes including numerous phospholipases (PL) A2, cyclooxygenases (COX)-1 and 2, and several newly discovered terminal PGE synthases. It is involved in important biological events such as female reproduction, neuronal function, inflammation, vascular hypertension, tumorigenesis, kidney function, and also plays key roles in inflammatory and neurologic disorders (Kobayashi et al., (2002) In vivo progestin treatments inhibit nitric oxide and endothelin-1-induced bovine endometrial prostaglandin (PG)E(PGE) secretion in vitro. Prostaglandin and Other Lipid Mediat; 68-69:557-574). PGE2 exerts its effects through four G-protein-coupled receptor subtypes known as EP1, EP2, EP3, and EP4, which it binds with similar affinity (Narumiya et al, (1999) Prostanoid receptors: structures, properties, and functions. Physiol Rev. 79:1193-1226).
The four different receptor subtypes results in diverse functional responses (Breyer et al., (2001) Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol. 41: 661-690). The EP1 receptor has been shown to mediate pro-algesic responses, since knockout mice studies result in reduced pain sensitivity (Stock et al., 2001) and EP1 antagonism reduces hyperalgesia and allodynia in the chronic constriction injury (CCI) model of neuropathic pain (Kawahara et al., 2001). Similarly, studies show that EP3 receptor antagonism is also analgesic (Hosoi et al., Prostaglandin E receptor EP3 subtype is involved in thermal hyperalgesia through its actions in the preoptic hypothalamus and the diagonal band of Broca in rats, Pain 71:303-311).
PGE2 signaling via the EP2 receptor has been linked to a proinflammatory and proamyloidogenic pathway towards the development of Alzheimer's disease (AD) pathology in a model of familial AD (Liang et al, (2005) Deletion of the prostaglandin E2 EP2 receptor reduces oxidative damage and amyloid burden in a model of Alzheimer's disease. J. Neurosci. 25(44):10180-7), while activation of EP2 and EP4 receptors has been shown to be involved in the in vivo production of Abeta in the pathogenesis of AD (Hoshino et al., (2007) Involvement of prostaglandin E2 in production of amyloid-beta peptides both in vitro and in vivo. J Biol. Chem. 282:32676-32688).
Recent studies have also implicated the EP4 receptor in proinflammatory responses. EP4 receptor signaling has been shown to be involved in increased inflammation and disease progression in rheumathoid arthritis (McCoy et al., (2002) The role of prostaglandin E2 receptors in the pathogenesis of rheumatoid arthritis, J. Clin. Invest. 110, pp. 651-658), while studies with EP4 receptor antagonists have demonstrated reduction of inflammatory pain in vitro and in animal models. Investigations with an EP4 antagonist as well as EP4 knock-down studies resulted in reduction of inflammation-induced thermal and mechanical behavioral hypersensitivity as well as sensitization of capsaicin-evoked currents in DRG neurons in vitro (Lin et al., (2006) Prostaglandin E2 receptor EP4 contributes to inflammatory pain hypersensitivity. J Pharmacol Exp Ther. 319:1096-103). In another study, EP4 receptor antagonists administered to adjuvant-induced arthritis rats reversed paw swelling to normal levels (Murase et al., (2008) Effect of prostanoid EP4 receptor antagonist, CJ-042,794, in rat models of pain and inflammation. Eur J. Pharmacol. 580:116-121). Thus, the EP4 receptor is a potential target for the pharmacological treatment of inflammation and pain.
A recent study has demonstrated that EP2-EP4 signaling promotes T helper (Th) 1 cell differentiation, and EP4 signaling is essential for IL-23 production in the expansion of Th17 cells (Yao et al., (2009) Prostaglandin E2-EP4 signaling promotes immune inflammation through Th1 cell differentiation and Th17 cell expansion. Nat. Med. 15:633-640). They showed that daily oral administration (twice per day) of an EP4 antagonist (ONO-AE3-208) was able to suppress the symptoms of an experimental mouse model exhibiting multiple sclerosis-like symptoms, namely, the experimental autoimmune encephalomyelitis (EAE) model.
Experimental autoimmune encephalomyelitis (EAE) is the most frequently used animal model for immune mediated effects of multiple sclerosis (MS), studying the progression of the demyelination of axons and test the efficacy of potential therapeutic effects by candidate compounds (Gold et al., (2006) Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain. 129:1953-1971).
In the active EAE model where myelin oligodendrocyte glycoprotein (MOG) is directly injected into an animal, a chronic progressive form of EAE is exhibited in response to the immunization. MOG is a transmembrane protein that is expressed on the surface of oligodendrocytes in the central nervous system. It is used as a target antigen in facilitating demyelination which leads to multiple sclerosis (MS) like symptoms that are observed in mice (Silber and Sharief, (1999) Axonal degeneration in the pathogenesis of multiple sclerosis. J. Neurol. Sci. 170:11-18). Upon introduction of MOG, symptomatic manifestation takes anywhere between 7 to 10 days. The clinical scores were given on a 9 point score scale based on the previously published literature (Stromnes and Goverman, (2008) Active induction of experimental allergic encephalomyelitis. Nat. Protoc. 1:1810-1818).
There are several molecular targets in the EAE model that must be studied in order to demonstrate the efficacy of candidate compounds in becoming a potential therapeutic agent for treating autoimmune diseases (Sloane et al., (2009) Anti-inflammatory cytokine gene therapy decreases sensory and motor dysfunction in experimental multiple sclerosis: MOG-EAE behavioral and anatomical symptom treatment with cytokine gene therapy. Brain, Behav. Immunity. 22:600-605). Cytokines are one such target where numerous studies showing an increase in myelinotoxic inflammatory cytokines such as interferon-γ (IFN-γ) coinciding with the active phase of EAE (Zaheer et al., (2007) Diminished cytokine and chemokine expression in the central nervous system of GMF-deficient mice with experimental autoimmune encephalomyelitis. Brain Res. 1144:239-247).
Cytokines are small secreted proteins which mediate and regulate immunity, inflammation, and hematopoiesis and are believed to be one of the key signaling molecules in the disease progression of EAE. They must be produced de novo in response to an immune stimulus. Autoimmune responses observed in EAE are believed to be mediated via helper T (Th) 1 pathway and interferon-gamma (IFN-γ) is the hallmark cytokine of the Th1 immune response (Muthian et al., (2006) 1,25 Dihydroxivitamin-D3 modulates JAK-STAT pathway in IL-12/INF-γ axis leading to Th1 response in experimental allergic encephalomyelitis. J. Neurosci. Res. 83:1299-1309). Therefore, being able to reduce the amount of cytokines such as IFN-γ is imperative in determining the effectiveness of compounds as potential therapeutic agents for the EAE model and ultimately in treating autoimmune diseases such as multiple sclerosis.
Recently, another distinct helper T subset termed Th17 pathway has been suggested as a long term mediator of autoimmune pathology (Narumiya et al., (2009) Prostaglandin E2-EP4 signaling promotes immune inflammation through Th1 cell differentiation and TH17 cell expansion. Nat. Med. 15:633-640). Previously, the T-cell mediated autoimmune responses seen in diseases such as EAE and MS were thought to be caused by Th1 pathway alone. However, recent evidence points toward much more intricate balances between Th1/Th2 and the newly discovered and designated Th17 pathways. It has been suggested that although the initial pathological responses are still initiated by the Th1 pathway, the sustained tissue damage typically observed in autoimmune diseases is mediated by the Th17 pathway (Steinman, (2007) A brief history of TH 17, the first major revision in the TH1/Th2 hypothesis of T cell-mediated tissue damage. Nat. Med. 13:139-145). The initiation of Th17 differentiation is believed to involve TGF-β and IL-6 which in turn activate orphan nuclear receptor RORγt (Korn et al., 2009 IL-17 and Th17 cells. Annu. Rev. Immunol. 27:485-517). RORγt is a transcriptional factor for IL-17 whose role in tissue inflammation and autoimmune response has been extensively documented previously.
Interleukin-6 (IL-6) is another key cytokine in the acute phase of the proinflammatory reaction in the immune response. IL-6 signals through a cell-surface type I cytokine receptor complex which in turn activates the JAK-STAT pathway (Khoury et al., (2005) Cytokines in multiple sclerosis: form bench to bedside. Pharm. Ther. 106:163-177). In addition, IL-6 is believed to be one of the co-affectors in T helper (Th) 17 differentiation along with transforming growth factor-β (TGF-β) and interleukin-23 (IL-23).
Another major molecular target is signal transducers and activators of transcription (STAT) protein pathway. STAT proteins are nuclear proteins involved in cell survival, differentiation, proinflammatory reactions, and cytokine signaling. It is a family of seven member proteins with each STAT protein activated by different cytokines. In addition, STAT proteins are a part of Janus (JAK)-STAT pathway, one of the main signaling pathways for cytokine and growth factors.
STAT3, upon activation by IL-6, translocates into the nucleus where it induces gene expression (Ihle. (2001) The Stat family in cytokine signaling. Curr. Opin. Cell Biol. 13:211-217). STAT4 is believed to be activated by IL-12 and the study of STAT4 knockout mice has been shown to have impaired Th1 response which is responsible for adaptive immunity (Bright et al., (2008) Stat 4 isoforms differentially regulate inflammation and demyelination in experimental allergic encephalomyelitis. J. Immun. 181:5681-5690). STAT6 is activated by IL-4 and IL-13 and the mice lacking STAT6 genes have been shown to have impaired Th2 response which is responsible for humoral immunity (Takeda and Akira, (2000) STAT family of transcription factors in cytokine-mediated biological responses. Cytokine & Growth Factor Rev. 11:199-207). STAT3, in particular, has been linked to autoimmune diseases such as multiple sclerosis and EAE where high levels of STAT 3 have been detected during the acute phase of EAE (Yang et al., STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J. Biol. Chem. 282:9358-9363).
Aside from chronic forms of inflammation such as autoimmune diseases, acute inflammation is one of the most basic defense mechanisms an organism uses. Acute inflammation is mainly initiated by macrophages and dendritic cells. Upon introduction of allergens, these immune cells mount proinflammatory response initiating cytokines such as tumor necrosis factor-α (TNF-α) and INF-β. There is also an increase in blood flow from vasodilation. This increase in blood flow results in the formation of edema and swelling around the site of the inflammation. Many potential immune-mediating substances are tested on in vivo models of endotoxin-induction of proinflammatory cytokines (Tang et al., (2007) LPS-induced TNF-alpha factor (LITAF)-deficient mice express reduced LPS-induced cytokine. Evidence for LITAF-dependent LPS signaling pathway. PNAS. 103:13777-13782) and allergen-induced edema formation (Tamura et al., (2004) Effects of olopatadine hydrochloride, an antihistamine drug, on skin inflammation induced by repeated topical application of oxazolone in mice. Br. J. Dermatol. 151(6):1133-1142).
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels located primarily within the central nervous system (CNS). They belong to the family of ionotropic glutamate receptors and exist as multiple subtypes due to the different combinations of subunits—NR1, NR2 (NR2A, NR2B, NR2C, NR2D) and NR3—that can be expressed. In addition to the agonist binding site, NMDA receptors have multiple distinct binding sites for various compounds that enhance, modulate and inhibit the activation of the receptors.
It is known that NMDA receptors are involved in neuronal communication and play important roles in synaptic plasticity and mechanisms that underlie learning and memory. Under normal conditions, NMDA receptors engage in synaptic transmission via the neurotransmitter glutamate, which regulates and refines synaptic growth and plasticity. However, when there are abnormally high levels of glutamate (i.e. under pathological conditions), NMDA receptors become over-activated, resulting in an excess of Ca2+ influx into neuronal cells, which in turn causes excitotoxicity and the activation of several signaling pathways that trigger neuronal apoptosis. Glutamate-induced apoptosis in brain tissue also accompanies oxidative stress resulting in loss of ATP, loss of mitochondrial membrane potential, and the release of reactive oxygen species and reactive nitrogen species (e.g. H2O2, NO, OONO−, O2−) causing associated cell damage and death. Decreased nerve cell function and neuronal cell death eventually occur. Excitotoxicity also occurs if the cell's energy metabolism is compromised.
Over-activation of the NMDA receptors is implicated in neurodegenerative diseases and other neuro-related conditions as it causes neuronal loss and cognitive impairment, and also plays a part in the final common pathway leading to neuronal injury in a variety of neurodegenerative disorders such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and Huntington's disease, as well as conditions such as stroke. Recent findings have implicated NMDA receptors in many other neurological disorders, such as multiple sclerosis, cerebral palsy (periventricular leukomalacia), and spinal cord injury, as well as in chronic and severe mood disorders (Mathew S J et al., Rev Bras Psiquiatr, 27:243-248 (2005)).
For instance, glutamate excitotoxicity has been linked to inflammatory autoimmune demyelination in MS. The disease pathology involves loss of myelin, oligodendrocytes and axons due to an inflammatory attack on the central nervous system. Several studies indicate the role of excessive glutamate in the pathology of MS (Flanagan et al. (1995) Neurotoxin quinolinic acid is selectively elevated in spinal cords of rats with experimental allergic encephalomyelitis. Journal of Neurochemistry; 64 (3): 1192-1196); Sarchielli et al., (2003) Excitatory amino acids and multiple sclerosis: evidence from cerebrospinal fluid. Archives of Neurology. 2003; 60(8):1082-1088).
Excessive glutamate levels have also been observed in the cerebrospinal fluid obtained from patients with MS (Stover et al. (1997) Neurotransmitters in cerebrospinal fluid reflect pathological activity. European Journal of Clinical Investigation. 27(12):1038-1043). Furthermore, investigations with NMDA receptor antagonists such as Memantine results in suppression of MS pathogenesis as well as improvement of neurovascular functions (Paul and Bolton (2002) Modulation of blood-brain barrier dysfunction and neurological deficits during acute experimental allergic encephalomyelitis by the N-methyl-D-aspartate receptor antagonist memantine. The Journal of Pharmacology and Experimental Therapeutics. 2002; 302(1):50-57). Thus, compounds designed to antagonise the actions of NMDA receptors could potentially offer therapeutic benefits to MS patients and are currently in development (Farrell et al. (2005) Emerging therapies in multiple sclerosis. Expert Opin Emerg Drugs. 2005; 10(4):797-816).
Melanocortins (MC) receptors belong to the class of G protein-coupled receptors. More specifically, they are a group of pituitary peptide hormones, which include the adrenocorticotropic hormone (ACTH) and the alpha, beta and gamma melanocyte-stimulating hormones (MSH). They are derived from the pro-hormone proopiomelanocortin (Adan et al., (2000) Melanocortins and the brain: from effects via receptors to drug targets. Eur J Pharmacol 405: 13-24). MCs act through a multitude of melanocortin receptors designated MC1 through MC5. MC1 receptors are expressed in macrophages and monocytes, keratinocytes and melanocytes, endothelial cells, glioma cells and astrocytes, and pituitary and periaqueductal grey matter, where they are involved in melanogenesis and anti-inflammatory processes (Kang et al., (2006) A selective small molecule agonist of the melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice. J Leukoc Biol 80: 897-904; and Slominski et al., (2004) Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84: 1155-228).
ACTH binds to the MC2 receptor (ACTH receptor) and is mainly expressed in the adrenal glands and the adrenal cortex. While MC3 is expressed in both periphery and neural tissues, MC4 is mainly found in the CNS and is the second neural MC receptor as they are expressed in multiple regions of the brain including the cortex, thalamus, hypothalamus, brainstem, and spinal cord. The receptor is also highly expressed in the paraventricular nucleus and is involved in the modulation of pituitary function. MC5, highly homologous to MC4, is the only MC receptor found in skeletal muscle. It is broadly expressed in peripheral tissue, while also present in specific brain regions.
MC4 receptor activity has been linked to neurite outgrowth and peripheral nerve regeneration (Tanabe et al., (2007) Melanocortin receptor 4 is induced in nerve-injured motor and sensory neurons of mouse. J Neurochem 101:1145-52; and Adan et al., (1996) Melanocortin receptors mediate and neuroprotection in brain ischemia stroke (Giuliani et al., 2006), and inflammatory responses in astrocytes (Caruso et al., (2007) Activation of melanocortin 4 receptors reduces the inflammatory response and prevents apoptosis induced by lipopolysaccharide and interferon-gamma in astrocytes. Endocrinology 148: 4918-26).
DA001 also induces CCAAT/enhancer binding protein beta (C/EBP-b) mRNA expression in cortical neuron cultures. C/EBP-b is an important transcriptional activator in the regulation of genes involved in immune and inflammatory responses. It specifically binds to an IL-1 response element in the IL-6 gene, and thought to play a role in the regulation of acute-phase reactions, inflammation and hemopoiesis. It is also involved in the differentiation process of various cell types including liver cells, adipoctyes and skin cells (reviewed in Sebastian and Johnson, (2006) Stop and go: anti-proliferative and mitogenic functions of the transcription factor C/EBPbeta. Cell Cycle 5(9):953-7; and Kalvakolanu and Roy, (2005) CCAAT/enhancer binding proteins and interferon signaling pathways. J Interferon Cytokine Res. 25(12):757-69). Homozygotes for targeted null mutations exhibit post-natal lethality and immature death. In neurons, it is involved in neurotrophin signaling and neuronal differentiation (Sterneck and Johnson, (1998) CCAAT/enhancer binding protein beta is a neuronal transcriptional regulator activated by nerve growth factor receptor signaling. J Neurochem. 70(6):2424-33; Menard et al., (2002) An Essential Role for a MEK-C/EBP Pathway during Growth Factor-Regulated Cortical Neurogenesis. Neuron 36, 597-610; and Cortés-Canteli et al., (2002) CCAAT/enhancer-binding protein beta plays a regulatory role in differentiation and apoptosis of neuroblastoma cells. J Biol. Chem. 277(7):5460-7).
Recently, a novel cell survival function for C/EBP-b has been reported. The activity of C/EBP-b is lost before the onset of cell death and the pathologic response in cortical neurons induced by hypoxia involves C/EBP-b-mediated survival signals (Halteman et al., (2008) Loss of c/EBP-beta activity promotes the adaptive to apoptotic switch in hypoxic cortical neurons. Mol Cell Neurosci. 38(2):125-37). On the other hand, C/EBP-b is also essential in response to neuronal injury by transcriptionally activated regeneration-associated gene expression (Nadeau et al., (2005) A transcriptional role for C/EBP beta in the neuronal response to axonal injury. Mol Cell Neurosci. 29(4):525-35). C/EPB-b may therefore play a role in DA001-mediated activities.
Therefore, there is a need to develop other effective receptor antagonists, such as NMDA, MC and PGE2 receptor antagonists that have high potency and are capable of preventing, treating and/or ameliorating inflammation and/or pain, central nervous system disorder and other diseases and conditions. The present invention satisfies this and other needs.